Abstract
BackgroundJapanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E) is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system.ResultsThe coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line.ConclusionThe Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.
Highlights
Japanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen
They are membrane vesicles of 20 nm in diameter containing JEV preM/M and E proteins embedded in the lipid bilayer, and showed similar behaviour to the slowly sedimenting hemagglutinin particles released from JEV-infected cells on sucrose density gradients [10,17]
Expression of JEV E protein in Schneider 2 (S2)/ZF-ZR and S2/JEF-JER cells Transfected S2 cells, either S2/ZF-ZR or S2/JEF-JER appeared normal morphologically and their growth was comparable with normal control S2 cells
Summary
Japanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen. For JEV, such particles were produced in mammalian cells infected with recombinant poxviruses encoding the signal sequence of preM, preM and E proteins, and were designated as the subviral extracellular particles (EPs). JEV preM and E proteins were expressed in Sf9 insect cells infected with recombinant baculovirus, and the intracellular E protein was shown to be protective in mice against lethal JEV challenges [21,22]. It is not known whether EPs were formed or not in this system
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